Congenital heart defects are the most common form of birth anomalies, affecting 6 to 8 patients per 1000 births, and are the leading non-infectious cause of death in children. Hypoplastic left heart syndrome is the most devastating of these conditions and is characterized by marked hypoplasia or atresia of the left ventricle as well as hypoplasia of the ascending aorta and aortic valve. Palliation of this disease can be achieved through either a staged reconstruction, converting the cardiovascular system to a single ventricle based pump, or cardiac transplantation. Both of these procedures carry significant risks with undetermined long-term effects.
Advances in fetal imaging have made possible the early gestational diagnosis of most anatomic congenital anomalies. Advances in prenatal intervention and fetal tissue sampling have resulted in consideration of the strategy of perinatal tissue engineering: i.e. the prenatal harvest, isolation, and in vitro expansion of autologous fetal cells for the purpose of engineering a tissue construct with subsequential surgical reconstruction immediately after birth. To date this concept has been explored as an experimental therapeutic strategy for reconstruction of muscular diaphragmatic defects, bladder exstrophy, as well as superficial skin defects.
With respect to adults, despite tremendous advances in the treatment of acute myocardial infarction, post-infarction congestive heart failure remains a difficult clinical problem. Coronary occlusion with consequent regional ischemia leads to loss of cardiomyocytes and progressive replacement of muscle by collagenous tissue resulting in a myocardial scar. Even a small scar progressively expands, directly affecting the contractile function of adjacent border zone myocardium and overall global contractile function. Thus, left ventricular dilatation and heart failure can occur even after a myocardial infarction of moderate size. While novel therapeutic strategies such as biventricular pacing, mitral valve repair and ventricular remodeling surgery may improve the quality of life in a portion of patients with end-stage heart failure, heart transplantation is still the only available treatment that significantly lengthens life expectancy. Transplantation, however, is limited by a chronic shortage of donor hearts.
Treatment by transplantation is also limited by the problem of rejection. Advances in immunosuppression have significantly improved the treatment of acute rejection but chronic rejection in the form of cardiac allograft vasculopathy remains a leading cause of death in transplant recipients. Organ rejection can be initiated by two separate pathways of allorecognition. Unlike physiologic “indirect” T cell mediated allorecognition, where a foreign antigen (in this case an alloantigen) is processed and presented by a recipient antigen presenting cell to a recipient T cell in an MHC self-restricted manner, “direct allorecognition” is driven by the recognition of the intact alloantigen on an allogeneic antigen presenting cell. Due to this molecular mimicry, the precursor frequency of alloreactive T cells activated by this pathway is several logs higher than those that can be activated by indirect allorecognition alone. The vigor of the initial episode of acute rejection after organ transplantation has been classically attributed to donor-derived hematopoietic antigen presenting cells, which migrate from the transplanted organ to the host's peripheral lymphoid tissue and activate T lymphocytes by the direct pathway of allorecognition. As these passenger leucocytes survive only briefly after migration to the host lymphoid system, allorecognition at later points and subsequential chronic rejection has been attributed to the weaker “indirect pathway”.
The present inventors have investigated the role of allograft parenchymal cells in direct allorecognition. (Kreisel, D., et al., A simple method for culturing mouse vascular endolthelium, J. of Immunological Methods, 254: 31-45 (2001); Krupnick, et al., Multiparameter flow cytometric approach for simultaneous evaluation of T lymphocyte-endothelial cell interactions, Cytometry (Communications in Clinical Cytometry) 46: 271-280 (2001)). It has been found that even in the absence of professional donor-derived hematopoietic antigen presenting cells, donor parenchymal cells, most likely vascular endothelial cells, can initiate direct alloantigen presentation to alloreactive host T lymphocytes. In fact, the presence of donor derived hematopoietic antigen presenting cells does not affect the tempo of CD8+T cell mediated allograft rejection which can be mediated entirely by parenchymal cells (Kreisel D, Krupnick A S, Gelman A E, Engels F H, Popma S H, Krasinskas A M, Balsara K R, Szeto W Y, Turka L A, Rosengard B R (2002), Non-hematopoietic allograft cells directly activate CD8+ T cells and trigger acute rejection: an alternative mechanism of allorecognition, Nature Medicine 8:233-239). Thus, a cell population that resides permanently in the transplanted organ and plays a critical role in organ function also can initiate a powerful pathway of antigen presentation leading to chronic rejection and organ destruction. This new paradigm of allorecognition presents yet another major barrier to successful, long-term, solid organ transplantation. These results call into question whether whole organ replacement is truly necessary for treatment of end stage heart failure. Since the majority of heart failure patients suffer a decline in function due to deleterious ventricular remodeling resulting from a limited size infarction, heart failure may be prevented by restoring contractility solely in this portion of the myocardium.
These issues have led the present inventors to reexamine the necessity of whole organ transplantation and to investigate the possibility that replacement of only the portion of myocardium lost to ischemic insult or congenital malformation might suffice in treating cardiac dysfunction. Despite long-standing assumptions that adult myocardium can not regenerate and lacks stem cells to support such regeneration, recent experimental data has shown that adult and fetal stem cells can differentiate into cardiac myocytes, repopulate myocardial scar, and improve myocardial function. Based on these findings experimental efforts have become focused on the establishment of small animal models of adult and congenital cardiac disorders in order to develop novel cell delivery strategies such as cellular cardiomyoplasty, and myocardial tissue engineering, as well as to harness the innate regenerative capacity of myocardial tissue.
Tissue engineering is a multidisciplinary field combining principles of engineering and biological science and focuses on creating viable organs and tissues to address the limitations of allogeneic solid organ and cell transplantation. The basic principle involves in vivo implantation of a cell-matrix construct in order to replace diseased or deficient tissues with recapitulation of structure after resorption or remodeling of the matrix. Recent developments in successful tissue engineering of cardiac valves and blood vessels have opened up the possibility of reconstructing the hypoplastic left heart by replacing the atretic aortic arch and valve with autologous living constructs that can properly grow during the child's development (Hoerstrup S P, Sodian R, Sperling J S, Vacanti J P, Mayer J E, Jr. (2000), New pulsatile bioreactor for in vitro formation of tissue engineered heart valves. Tissue Engineering 6:75-79; Niklason L E, Gao J, Abbott W M, Hirschi K K, Houser S, Marini R, Langer R (1999), Functional arteries grown in vitro. Science 284: 489-493; Shinoka T, Breuer C K, Tanel R E, Zund G, Miura T, Ma P X, Langer R, Vacanti J P, Mayer J E, Jr. (1995), Tissue engineering heart valves: valve leaflet replacement study in a lamb model, Annals of Thoracic Surgery 60: S513-516). Complete correction of cardiac anomalies, however, is limited by the inability to engineer functional ventricular tissue and to enlarge the ventricular cavity. Manipulation of the native heart for tissue engineering purposes is possible in a large animal model under complete cardiopulmonary bypass, but no comparable technology has been available for rodent models (Shinoka T, Ma P X, Shum-Tim D, et al., Tissue-engineered heart valves, Autologous valve leaflet replacement study in a lamb model. Circulation 1996; 94: II164-168). Cardiac tissue engineering in a murine model has been limited by the difficulty of achieving cardiopulmonary bypass utilizing standard techniques. This is unfortunate as the murine model offers the ideal opportunity to study in vivo myocardial tissue engineering with a substantial amount of in vitro data describing the construction of myocardial tissue already available (Carrier R L, Papadaki M, Rupnick M, Schoen F J, Bursac N, Langer R, Freed L E, Vunjak-Novakovic G (1999), Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization. Biotechnology & Bioengineering 64: 580-589; Zimmermann W H, Fink C, Kralisch D, Remmers U, Weil J, Eschenhagen T (2000), Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes. Biotechnology & Bioengineering 68: 106-114). A rodent model also offers numerous other advantages such as the availability of inbred syngeneic strains, a defined system of stem cells for transplantation, as well as decreased animal cost (Prockop D J. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997; 276: 71-74).
The present inventors have developed a novel small animal model of ventricular tissue engineering utilizing heterotopic heart transplantation. Rat heterotopic heart transplantation offers the ability to operate on an explanted donor organ without the necessity of cardiopulmonary bypass or cardiovascular compromise of the recipient host. By manipulating the microsurgical anastomoses at the time of reimplantation, it is possible to vary the hemodynamic loading of the left ventricle. The present inventors have tested several biocompatible matrices for ventricular replacement, validated the ability to augment ventricular volume in a functioning heart, and provided evidence for myocardial replacement utilizing three-dimensional, stem cell-seeded patches or scaffoldings in accordance with the present invention.
Since seeding stem cells or myoblasts into the compromised ventricle has been shown to improve cardiac function, numerous laboratories have focused on studying cellular cardiomyoplasty as a means of reversing myocardial dysfunction (Sakai T, Li R K, Weisel R D, et al. Autologous heart cell transplantation improves cardiac function after myocardial injury. Annals of Thoracic Surgery 1999; 68: 2074-2080; discussion 2080-2071; Li R K, Jia Z Q, Weisel R D, Merante F, Mickle D A. Smooth muscle cell transplantation into myocardial scar tissue improves heart function, Journal of Molecular & Cellular Cardiology 1999, 31: 513-522; Klug M G, Soonpaa M H, Koh G Y, Field L J, Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts, Journal of Clinical Investigation 1996; 98: 216-224). This technology, however, does not address the absence of myocardium resulting from congenital malformation. The paucity of investigation within this field has been mainly due to the lack of a suitable animal model for the study of ventricular tissue engineering.
The present inventors have provided a model for ventricular tissue engineering that can facilitate studies into the replacement or augmentation of myocardial tissue. By avoiding the need for cardiopulmonary bypass, this model can be utilized in small laboratory animals. By avoiding manipulation and infarction of the native heart excessive animal loss is prevented. Since the described model allows the creation of a fully functioning or an unloaded left ventricle, tissue engineering and remodeling can be studied under normal physiologic conditions or under conditions modeling mechanical circulatory support with a left ventricular assist device.
Composite tissue constructed from biocompatible and biodegradable scaffoldings seeded with single cells or tissue equivalent has been investigated for use in tissue engineering (Shinoka T, Ma P X, Shum-Tim D, et al, Tissue-engineered heart valves, Autologous valve leaflet replacement study in a lamb model, Circulation 1996; 94: II164-168; Sakata J, Vacanti C A, Schloo B, Healy G B, Langer R, Vacanti J P, Tracheal composites tissue engineered from chondrocytes, tracheal epithelial cells, and synthetic degradable scaffolding, Transplantation Proceedings 1994; 26: 3309-3310; Kaihara S, Vacanti J P, Tissue engineering: Toward new solutions for transplantation and reconstructive surgery, Archives of Surgery 1999 134: 1184-1188; Juang J H, Bonner-Weir S, Ogawa Y, Vacanti J P, Weir G C, Outcome of subcutaneous islet transplantation improved by polymer device, Transplantation 1996; 61: 1557-1561). Little data, however, is available on the use of such materials for ventricular replacement. The in vitro construction of porous hydrogels with similar characteristics to myocardial extracellular matrix has been previously reported. When cardiac myocytes are cultured within this matrix they organize into a three-dimensional myocardial tissue with physiologic characteristics similar to those of native heart tissue (Zimmermann W H, Fink C, Kralisch D, Remmers U, Weil J, Eschenhagen T, Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes. Biotechnology & Bioengineering 2000; 68: 106-114).
However, there remains a need for the in vitro construction and in vivo transplantation of three dimensional tissue. The present inventors demonstrate herein that mesenchymal stem cells, seeded on a three-dimensional matrix, or cell carrier, can engraft and differentiate within the left ventricle of the heart. A delivery vehicle, or patch, for delivering such cells is described herein.
Macroporous scaffoldings fabricated from polymers of lactide (PLLA) and glycolide (PGA) have been studied as an alternative to naturally derived scaffoldings. They break down by simple hydrolysis to natural metabolic products and their highly porous characteristics (95% porosity) allow the delivery and polymerization of the transplanted cells and collagen hydrogel within the interstices of the matrix (Mooney D J, Breuer C K, McNamara K, Vacanti J P, Langer R, Fabricating tubular devices from polymers of lactic and glycolic acid for tissue engineering. Tissue Engineering 1995; 1: 107-118). PGA based scaffoldings have been particularly attractive to the field of tissue engineering due to their rapid rate of degradation and near complete hydrolysis by four weeks in vivo (Peters M C, Mooney D J. Synthetic extracellular matrices for cell transplantation, In: Liu D M, Dixit V, eds., Materials Science Forum Vol. 250. Switzerland: Trans Tech Publications, 1997: 43-52). PGA mesh has been successfully used for the delivery of chondrocytes and tracheal epithelial cells for engineering of cartilage; however, its utility for myocardial tissue engineering has not been successfully demonstrated (Sakata J, Vacanti C A, Schloo B, Healy G B, Langer R, Vacanti J P, Tracheal composites tissue engineered from chondrocytes, tracheal epithelial cells, and synthetic degradable scaffolding, Transplantation Proceedings 1994; 26: 3309-3310). Studies by the present inventors indicate that the use of this matrix within the heart results in an intense inflammatory response. Since this inflammatory response has limited PGA's ability to serve as a vehicle for cellular transplantation in other organ systems, similar limitations will likely be encountered in the heart.
As will be explained below, the present inventors have successfully demonstrated the use of PLLA matrix for cell transplantation in the heart. Although little degradation of PLLA occurs within the first year, with the potential for infectious complications associated with any foreign body, minimal inflammation was detected by the present inventors in scaffoldings constructed around PLLA matrix.
Consideration of the cell delivery approach for broader based applications is limited by the variety of cells necessary for proper reconstruction of extensive congenital malformations involving several organ systems or anatomic defects requiring a variety of tissues for reconstruction, such as congenital cardiac anomalies. The application of this concept to cardiac tissue engineering has been further complicated by the practical concerns of donor site morbidity and the difficulty of expanding differentiated cardiac myocytes in vitro. These concerns may be overcome by the ability to isolate and expand a population of multilineage stem cells.
Mesenchymal stem cells can be isolated from the bone marrow of numerous species and have been shown under specific circumstances to differentiate into various cell types including osteocytes, chondrocytes, adipocytes, as well as skeletal and cardiac myocytes (Pittenger M F, Mackay A M, Beck S C, et al, Multilineage potential of adult human mesenchymal stem cells, Science 1999, 284 (5411): 143; Wakitani S, Saito T, Caplan A I, Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine, Muscle & Nerve 1995; 18 (12): 1417; Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro, Journal of Clinical Investigation 1999; 103 (5): 697). The feasibility of isolating such cells and expanding them ex vivo for autologous tissue engineering in the adult has previously been studied (Bruder S P, Kraus K H, Goldberg V M, Kadiyala S., The effect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects, Journal of Bone & Joint Surgery—American Volume 1998; 80 (7): 985).
As most anatomic defects, including those of the heart, are routinely diagnosed during the first trimester of gestation by prenatal ultrasonography, a recently described concept of perinatal tissue engineering has emerged. This therapeutic strategy involves in utero harvest of fetal tissue, isolation, in vitro expansion, and organization of such autologous cells on a synthetic matrix creating an engineered tissue construct. As all manipulation occurs parallel to ongoing gestation, at birth the infant can benefit from surgical reconstruction utilizing this autologous tissue. To date, this concept has been explored as an experimental therapeutic strategy for reconstruction of muscular diaphragmatic defects, bladder extrophy, as well as superficial skin defects. Due to donor site morbidity and the difficulty of in vitro expansion of differentiated cardiac myocytes, such an approach has not been applied to myocardial tissue engineering. As numerous investigators have described cardiomyocytic differentiation potential of adult bone marrow-derived mesenchymal stem cells, the present inventors hypothesized that the fetal liver, the hematopoietic organ in utero, might contain a similar population of cells. As described below, the present inventors have demonstrated that the fetal liver provides a rich source of mesenchymal stem cells.
The ovine model has been extensively utilized for studies of fetal surgery and perinatal tissue engineering with several established experimental models of human disease including congenital cardiac defects (Fishman N H, Hof R B, Rudolph A M, Heymann M A, Models of congenital heart disease in fetal lambs, Circulation 1978; 58 (2): 354). Comparable size of the human and ovine fetus, as well as minimal uterine irritability further increases the utility of this model for fetal studies. The present inventors have demonstrated herein that the ovine fetal liver is a potential source of multilineage mesenchymal stem cells that may be utilized for autologous perinatal tissue engineering, particularly of the heart.
Post-infarction congestive heart failure in adults and congenital heart defects in children remain serious health issues. While cardiac transplantation has been shown to prolong life expectancy, this current form of treatment is limited by lack of suitable organs and chronic rejection. The present inventors have demonstrated the use of engineered constructs to repair damage to the heart, without using whole organ transplantation. It is desirable to provide patches for use in such procedures to repair damage to the heart by, e.g., increasing ventricular volume. It is further desirable to use such patches as cell carriers that may be used to deliver to the heart stem cells for replacement of discrete areas of the myocardium. The present invention addresses these needs in the art.